Eco-friendly simultaneous multi-spectrophotometric estimation of the newly approved drug combination of celecoxib and tramadol hydrochloride tablets in its dosage form

Food and Drug Administration (FDA) recently approved co-formulated celecoxib and tramadol for the treatment of acute pain in adults. Three spectrophotometric methods were efficiently applied to estimate the co-formulated Celecoxib and Tramadol in their tablets; second derivative 2D-spectrophotometry technique (method I), induced dual-wavelength technique (method II) and dual-wavelength resolution technique (method III). The proposed methods were successfully validated following the International Council for Harmonisation (ICH) guidelines and statistically assessed based on the correlation coefficients, relative standard deviations as well as detection and quantitation limits. The obtained results revealed non-significant differences compared to the reported results as revealed by the variance ratio F test and Student t test. Moreover, the applied techniques were further assessed concerning their greenness based on the analytical eco-scale method revealing an excellent green scale with a final score of 95. The proposed spectrophotometric techniques could be applied for the routine analysis and quality control of the studied drugs in their dosage form.


Theoretical background
Method I: induced dual wavelength method (IDW). This method is intended for application to a binary mixture (X and Y) having completely overlapped zero-order absorption spectra at two wavelengths λ 1 and λ 2 respectively, in which the absorbance of the interfering substances at the selected wavelengths are not equal (so that absorbance difference at the said wavelengths is not equal to zero), rendering the conventional dual-wavelength method inapplicable 51 . The technique is relatively new and was utilized a few times to resolve some challenging components in mixtures 5,[51][52][53][54][55][56][57] . Briefly, the equations describing this situation are as follows: where A 1 is the mixture absorbance at λ 1 which is chosen as the λ max of X. A 2 is the mixture absorbance at λ 2 which is any other wavelength. An equality factor is calculated for component Y to eliminate its effect at the two wavelengths as follows: Substitution in Eq. (1) gives: (1) www.nature.com/scientificreports/ Then multiplying Eq. (2) by the equality factor F Y gives: Subtraction of Eq. (4) from Eq. (3) gives: The inspection of Eq. (5) indicates the sole dependence of the absorbance difference of the mixture on the C X with no interference from C Y . Thus, the concentration of component X can be computed from the following regression equation: Via plotting the absorbance difference values of the zero-order spectra of pure X at the selected wavelengths (∆A = A 1 − F Y A 2 ) versus the corresponding concentrations of X, the corresponding regression equation can be obtained.
Method II: dual wavelength resolution technique (DWRT). This method is applied when the IDW method is inapplicable to determine the concentration of the other component Y 51 . After the calculation of the concentration of the 1st component X using the IDW technique, the zero-order spectrum of component X is acquired by multiplication of the calculated concentration by the normalized absorptivity curve of component Y. The normalized absorptivity curve of component X is obtained by the division of the whole spectrum of X by its corresponding concentration giving a spectrum indicating the absorptivity of the analyte of interest (a X ) against all the measured wavelengths. The next step is obtaining the spectrum of component Y by subtracting the calculated spectrum of X from the spectrum of the corresponding mixture. Due to the problem of obtaining indefinite peaks in the zero-order spectrum of Y, the first derivative spectrum was obtained as a result and the corresponding regression equation was computed.
Method III: 2nd derivative technique. The 2nd derivative spectrophotometry was successfully applied for the simultaneous estimation of both drugs without a prior separation step. This method has the advantage of the evaluation of complex mixtures without chemical treatment. However, difficulties may sometimes arise as the signal-to-noise ratio can be reduced, in addition to the complex profiles obtained 58 .

Experimental
Apparatus. A Shimadzu ultraviolet-visible (UV-Vis) 1601 recording Spectrophotometer (P/N 206-67001, Procedure. Spectral characteristics. Working solutions of celecoxib and tramadol were prepared separately by transferring appropriate volumes from corresponding stock solutions in 10 mL volumetric flasks. The volumes were completed to the mark using distilled water as a solvent. The blank used was distilled water and methanol in the ratio 1:1. The zero-order spectra of each compound were measured within the range 190-500 nm and stored in the computer. Application of second derivative spectrophotometry. Series of different concentrations (2, 2.5, 3, 5, 7, 9, 12, 17, and 20 μg/mL) of celecoxib were prepared by transferring different volumes from stock solution into 10 mL volumetric flasks and performing dilution with distilled water to the mark. Similarly, a series of different concentrations (5,7,12,15,17,25,30,35,40,45, and 50 μg/mL) of tramadol HCl were prepared using distilled water for dilution. The absorption spectra of each concentration were measured against a blank consisting of distilled water and methanol in the ratio of 1:1 and then were stored in the computer. Then, each spectrum was smoothed (∆λ = 4 nm) and the second derivative of each spectrum was obtained (∆λ = 4 nm, scaling factor = 100). The cho- www.nature.com/scientificreports/ sen wavelengths were 269.45 nm and 295.58 nm for tramadol and celecoxib, respectively. The calibration curve was constructed by plotting the amplitudes at the chosen wavelengths versus the corresponding concentrations and the regression equation was subsequently obtained.
Application of induced dual wavelength (IDW) technique. Series of concentrations (6, 8, 9, 10, 11.5, 13, 14, 15, 17, 18, and 20 μg/mL) were prepared as previously described in accordance with the ratio of celecoxib in dosage forms (5.5 tramadol: 7 celecoxib). The absorbance at wavelengths 250 nm and 271 nm was recorded. The equality factor required to diminish the effect of Tramadol in the mixture was calculated by measuring the absorbance of the corresponding tramadol HCl concentrations at the same wavelengths and dividing the first absorbance reading (250 nm) by the second one (271 nm). Afterward, the absorbance of celecoxib at 271 nm was multiplied by this equality factor, and the result was subtracted from the absorbance at 250 nm to obtain ∆A. The calibration curve was constructed by plotting ∆A against the corresponding concentration and the regression equation was computed.
Application of dual wavelength resolution technique (DWR). Firstly, the normalized absorptivity curve for celecoxib was calculated by dividing the absorption spectrum of the drug by its corresponding concentration. A number of 11 normalized absorptivity curves was obtained and the average absorptivity curve was calculated. Secondly, the calculated absorption spectrum of celecoxib was obtained (after calculation of the concentration using the previous method) by multiplication of the calculated concentration of celecoxib by its average normalized absorptivity curve. After that, the corresponding absorption spectrum of tramadol was obtained by subtracting the calculated spectrum of celecoxib from the whole spectrum of the mixture. Finally, the obtained spectrum was smoothed (∆λ = 4 nm) and the first derivative was obtained (∆λ = 4 nm, scaling factor = 100). The absorbance of tramadol was then measured at 281.68 nm. The calibration curve was constructed by plotting the amplitudes at the chosen wavelengths against the corresponding concentrations and the regression equation was obtained.
Preparation of synthetic mixtures. A number of 11 mixtures was prepared with concentration ratios in the range of 5-15 μg/mL and 6-20 μg/mL for Tramadol and Celecoxib, respectively for the construction of calibration curves for both the Induced dual wavelength (IDW) and Dual wavelength resolution (DWRT) techniques. Laboratory-prepared tablets. Since the dosage form is not yet available in the Egyptian market, the laboratoryprepared tablet was used. Formula per tablet was prepared by weighing 56 mg of celecoxib, 44 mg of tramadol HCl, talc, starch, magnesium stearate, gelatin, and Avicel pH 112 FMC. The ingredients were triturated in a porcelain mortar and then transferred to a 100 mL volumetric flask. Around 60 mL of methanol was added.
After sonication for 30 min, the volume was completed to 100 mL with the same solvent. The solution was subsequently double-filtered using Whatmann no. 1 filter paper. Aliquot volumes were transferred to 10 mL measuring flasks and completed to the mark with distilled water to reach the following concentration ratios (7:9, 9:11.5, and 10:13 tramadol to celecoxib, respectively).
Evaluation of the method greenness. The method greenness was assessed based on the analytical Eco-scale approach which utilizes attributing penalty points to parameters that don't abide by the ideal green analysis. The analytical eco-scale was obtained using the following equation 49 :

Results and discussion
The completely overlapped spectra of the two drugs represented a challenge for their estimation (Fig. 2), hampering the use of other conventional methods such as the ratio spectra, ratio derivative, etc. The IDW technique was successfully applied to resolve celecoxib in the mixture of interest (Fig. 3).
The DWRT was used for the estimation of tramadol as the IDW technique was inapplicable (Fig. 4). The 2nd derivative technique was successfully applied for estimation of both drugs in their binary mixtures (Figs. 5, 6).
Optimization of experimental conditions. Effect of various solvents. Different solvents such as methanol, ethanol, and acetonitrile in addition to 0.1N HCl and 0.1N NaOH were used. No significant difference was found from spectra obtained in water, so distilled water was the solvent of choice due to its availability, cheap cost, and environmental safety.
Analytical performance and method validation. The methods were validated in accordance with the ICH guidelines 59 . The data indicated that the methods were of acceptable accuracy, precision, and specificity over the specified linearity range.
Analytical Eco-Scale = 100 − total penalty points. www.nature.com/scientificreports/ Linearity and range. The linear range as well as the correlation coefficient are calculated and the obtained results are summarized in Table 1. The linearity was evaluated by the least squares method indicating acceptable accuracy in accordance with ICH guidelines 59 .

Limits of detection (LOD) and limits of quantification (LOQ).
The Limit of Detection (LOD) and limit of quantification (LOQ) for each method were mathematically computed following ICH guidelines 59 and the data obtained are abridged in Table 1. The LOD was calculated from the following equation: While the LOQ was calculated from the following equation: where σ is the standard deviation of the response and s is the slope of the calibration curve estimated from the regression line. The ICH guidelines mandate the signal-to-noise ratio to be equal to 3:1 and 10:1 in the case of LOD and LOQ, respectively 59 .  www.nature.com/scientificreports/ Precision and accuracy. For the estimation of accuracy, the results of the proposed methods were compared to those obtained from previously reported methods following the guidelines of ICH using three replicate results for three different concentrations within the linear range 59 . For tramadol HCl, it was determined in methanol through its zero order spectra at λ max = 275 nm 60 , whereas for celecoxib, it was determined in 0.1N NaOH as solvent through its 1st order spectra at λ max = 269 nm 61 . These methods were also applied to the dosage forms and no significant difference was found as per the values of t test and F values 62 (Table 2). Intra and Inter day precision were studied by applying the proposed methods for the determination of three concentrations within the studied ranges on three successive times or three successive days in accordance with the ICH guidelines 59 . (Table 3).
Robustness. Distilled water was used for simplicity throughout the determination. No significant change was found when using different pH such as 0.1N NaOH or 0.1N HCl, so no buffer was used during the procedure. The robustness of the experimental procedure is thus confirmed as it was proven reliable regarding deliberate variations in parameters in agreement with ICH guidelines 59 .
Selectivity. Using the 2nd derivative technique, celecoxib was determined at λ max = 295.58 nm with no interference from tramadol HCl. Likewise, tramadol was determined at λ max = 269.45 nm with no interference from celecoxib, indicating acceptable Selectivity. Furthermore, Using the IDW technique, the selectivity was enhanced   www.nature.com/scientificreports/ and the interference from tramadol HCl was completely diminished by the equality factor, leading to the successful determination of celecoxib. Additionally, tramadol was successfully determined using DWRT with no interference from celecoxib. The excipients added to the laboratory-prepared tablets showed no interference with the applied techniques. Based on these findings, these methods provide good selectivity for the simultaneous determination of both drugs in a synthetic mixture and prepared tablet in the presence of excipients according to the requirements of the ICH 59 .
Application on laboratory prepared tablets. Table 4 shows the data of the proposed methods applied to the laboratory-prepared tablet. The results indicate that the proposed methods have acceptable accuracy and precision with respect to both drugs under investigation. In addition, no significant interference from the excipients in the prepared tablets was found, indicating acceptable selectivity for both drugs in the laboratory-prepared tablets.
Evaluation of the method greenness according to the analytical eco-scale approach. The concept of green chemistry aims at reducing both using and generating toxic hazardous materials. However, certain difficulties might arise upon evaluating the analytical methodologies such as the number of analytes to be determined, the techniques to be used as well as some important parameters needed to be considered such as LOD. In addition, the least green phase in the analytical process is sample preparation according to previously reported data 49 . Table 5 estimates the final score to be 95, indicating excellent green analysis techniques.

Conclusion
The goal of this study was to implement three Eco-friendly spectrophotometric methods for the assessment of the synthetic mixtures in addition to laboratory-prepared tablets of the recently approved drug combination (celecoxib/tramadol HCl). The applied methods including; the second derivative 2D-spectrophotometry technique (method I), induced dual-wavelength technique (method II), and dual-wavelength resolution technique (method III) revealed acceptable accuracy, good linearity, reproducibility as well as precision and can be applied for the routine analysis and quality control of the co-formulated mixture. Further, the methods also have the additional advantages of speed and simplicity, in addition to environmental safety as they don't require the use of hazardous solvents or sophisticated instruments. Statistical analysis has been accomplished illustrating non-significant differences compared to the previously reported data. Furthermore, the greenness index of the applied methods was measured via Eco-scale metric revealing an excellent green index of the methods employed. The proposed study poses simple, rapid, and reliable methods for the analytical assessment of the newly approved mixture besides being Eco-friendly. www.nature.com/scientificreports/ www.nature.com/scientificreports/

Data availability
All data generated or analyzed during this study are included in this published article.